Phosphorylation by liver glucokinase of D-glucose anomers at anomeric equilibrium

The relative contribution of each anomer of D-glucose to the overall phosphorylation rate of the hexose tested at anomeric equilibrium was examined in rat liver postmicrosomal supernatants under conditions aimed at characterizing the activity of glucokinase, with negligible interference of either hexokinase, N-acetyl-D-glucosamine kinase or glucose-6-phosphatase (acting as a phosphotransferase). Both at 10 degrees and 30 degrees C, the relative contribution of each anomer was unaffected by the concentration of D-glucose. At both temperatures, the alpha/beta ratio for the contribution of each anomer was slightly, but significantly, lower than the alpha/beta ratio of anomer concentrations. These findings, which are consistent with the anomeric specificity of glucokinase in terms of affinity, cooperativity and maximal velocity, reveal that the preferred alpha-anomeric substrate for both glycogen synthesis and glycolysis is generated by glucokinase at a lower rate than is beta-D-glucose-6-phosphate.     

Alpha melanocyte stimulating hormone inhibits immunostimulatory and inflammatory actions of interleukin 1

The ability of interleukin 1 (IL 1) to augment the proliferation of murine thymocytes in vitro was inhibited in a dose-dependent manner by the neuropeptide alpha-melanocyte-stimulating hormone (alpha MSH). The minimal effective concentration of alpha MSH was 10(-11) M. Maximal effect occurred between 10(-8) and 10(-7) M, with diminishing effectiveness at higher concentrations. IL 1-induced production of prostaglandin E (PGE) by fibroblasts was also inhibited by alpha MSH with a biphasic dose response. The minimal effective concentration was 10(-11) M, and maximum effect was achieved at 10(-10) M. alpha MSH appeared to affect the interaction of IL 1 with its target cells in a specific manner, because it did not inhibit basal mitogen-induced thymocyte proliferation or IL 2-induced proliferation of a cytotoxic T lymphocyte line. Furthermore, production of IL 1 by endotoxin-stimulated monocytes was not affected by alpha MSH. An analog of alpha MSH (Nle4, D-Phe7 alpha MSH), which is highly potent in other melanotropin-sensitive systems, did not affect the action of IL 1 on thymocytes, suggesting that the immunomodulatory effects of alpha MSH may not be mediated by the classic melanocyte alpha MSH receptor. The influence of alpha MSH on thymocytes and fibroblasts suggests that alpha MSH is an endogenous antagonist of IL 1, perhaps important for limiting inflammatory damage to host tissues.     

Temperature-sensitive expression of all-Torpedo and Torpedo-rat hybrid AChR in mammalian muscle cells

When the four subunits of the Torpedo californica nicotinic acetylcholine receptor (AChR) are expressed in mammalian fibroblasts, they properly assembly into alpha 2 beta gamma delta pentamers only at temperatures lower than 37 degrees C (Claudio, T., W. N. Green, D. S. Hartman, D. Hayden, H. L. Paulson, F. J. Sigworth, S. M. Sine, and A. Swedlund. 1987. Science (Wash. DC). 238:1688-1694). Experiments here with rat L6 myoblast cell lines indicate that this temperature sensitivity is not specific to fibroblasts, but is intrinsic to Torpedo subunits. A clonal isolate of L6 cells cotransfected with the four Torpedo subunit cDNAs synthesizes the exogenous AChR subunits at 37 degrees and 26 degrees C, but expresses Torpedo AChR complexes only at the lower temperature. When Torpedo alpha alone is expressed in L6 myotubes, hybrid AChRs are formed, again only at temperatures below 37 degrees C. These hybrid AChRs can contain either two Torpedo alpha subunits or one each of rat and Torpedo alpha, proving that the two alpha subunits in an AChR pentamer need not derive from the same polysome. Further analysis of hybrid and all-Torpedo AChR established that there is no internally sequestered pool of AChR at the nonpermissive temperature, and that the AChR, once formed, is thermostable. Two lines of experimentation with alpha subunits expressed in fibroblasts indicate that alpha polypeptides exhibit different conformations at 26 degrees and 37 degrees C, favoring the hypothesis that the temperature-sensitive step occurs before assembly and reflects, at least in part, misfolding of subunits: at 37 degrees C, there is a reduction in the fraction of alpha subunits that (a) bind the AChR antagonist alpha-bungarotoxin with high affinity; and (b) bind a monoclonal antibody that recognizes correctly folded and/or assembled alpha subunit.     

The immunolocalization of ACTH and alpha MSH in human and rat pituitaries.

Human and rat pituitaries were investigated immunohistochemically for ACTH and alpha MSH activity by means of the enzyme-labeling technique. In rat pituitaries cells present in both the anterior and intermediate lobes were reactive with the anti-ACTH antibodies, the cells from the intermediate lobe were also reactive with anti-alpha MSH antibodies. In human pituitaries, ACTH-immunoreactivity was found in cells from the anterior lobe and cells invading the posterior lobe. In 5 out of 15 pituitaries ACTH-immunoreactive cells located at or invading the posterior lobe were also reactive with the anti-alpha MSH antibodies. It is concluded that the human pituitary cells that invade the posterior lobe represent a population which is at least immunohistochemically identical with the intermediate lobe cells of the rat. The ACTH-immunoreactivity of intermediate lobe cells may be explained by the presence of a corticotropin-like intermediate lobe peptide (CLIP) which has been suggested to be a prohormonal fragment of alpha MSH.     

Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function

An autosomal dominant congenital cataract in human is associated with mutation of Arg-116 to Cys (R116C) in alpha A-crystallin. To investigate the molecular basis of cataract formation, rat alpha A-crystallin cDNA was cloned into pET-23d(+), and the site-directed mutants S142C (similar to wild-type human alpha A) and R116C/S142C or R116C (similar to human R116C variant) were generated. These were expressed in E. coli and the recombinant alpha A-crystallins purified by Sephacryl size-exclusion chromatography. The chaperone-like function of mutant R116C determined at 37 degrees C with insulin and alcohol dehydrogenase as target proteins was about 40% lower than those of wild-type and mutant S142C. Based on size-exclusion chromatography data, the oligomeric size of the R116C mutant was about 2000 kDa at 25 degrees C, 1400 kDa at 37 degrees C, and 900 kDa at 45 degrees C. In comparison, alpha A-wild-type and alpha A-S142C ranged from 477 to 581 kDa. Heat stability studies corroborated the effect of temperature on the dynamic quaternary structure of the R116C mutant. Circular dichroism spectra showed secondary and tertiary structural changes, and ANS fluorescence spectra showed loss of surface hydrophobicity in the R116C mutant. These findings suggest that the molecular basis for the congenital cataract with the alpha A-R116C mutation is due to the generation of a highly oligomerized alpha A-crystallin having a modified structure and decreased chaperone-like function.     

Effects of alterations in the activity of tuberohypophysial dopaminergic neurons on the secretion of alpha-melanocyte stimulating hormone

Administration of gamma-butyrolactone (GBL), an anesthetic which reduces dopaminergic neuronal activity, decreased the concentration of the dopamine (DA) metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the intermediate lobe of the pituitary gland, and increased alpha-melanocyte stimulating hormone (alpha MSH) concentrations in the serum of male rats. Bilateral electrical stimulation of the rostral arcuate nucleus, which contains perikarya of tuberohypophysial DA neurons, increased DOPAC concentrations in the intermediate lobe and decreased alpha MSH concentrations in the serum of GBL-anesthetized rats. Administration of the DA antagonist haloperidol prevented the decline in serum alpha MSH levels following arcuate nucleus stimulation, but had no effect on serum alpha MSH concentrations in sham-stimulated GBL-treated rats. These results indicate that GBL-induced decreases or stimulation-induced increases in the activity of tuberohypophysial DA neurons are accompanied by corresponding changes in the metabolism of DA in the intermediate lobe of the rat pituitary gland, and by reciprocal changes in the secretion of alpha MSH.     

Clots of beta-fibrin. Viscoelastic properties, temperature dependence of elasticity, and interaction with fibrinogen-binding tetrapeptides.

Clots of human beta-fibrin, in which only (or predominantly) the B fibrinopeptide is released, were formed at 14 degrees C by copperhead venom procoagulant enzyme (CVE or venzyme), at pH 8.5, ionic strength 0.45. The shear modulus of elasticity increased slowly and after several days attained a constant value, which was lower than those of alpha-fibrin or alpha beta-fibrin under the same conditions. Before studying the temperature dependence of elasticity, the CVE was then inhibited by introducing phenyl methyl sulfonyl chloride (PMSF) by diffusion. With increasing temperature, the modulus decreased progressively from 5 degrees C to nearly zero at 35 degrees and was essentially reversible with temperature change; recovery of elasticity after change from 34.5 degrees to 14 degrees required approximately 2 d but was considerably faster than the initial buildup of elasticity by CVE at 14 degrees. Creep and creep recovery measurements on unligated clots showed creep rates and irrecoverable deformation that were similar in magnitude to those of alpha-fibrin clots formed with batroxobin and much larger than those of alpha beta-fibrin clots formed with thrombin, under the same conditions. During creep and creep recovery, the differential modulus or compliance remained constant, showing that there was no permanent structural damage, and if network strands are severed in slow flow, they must rejoin in new configurations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of proline residues on parvalbumin stability

Despite its higher net charge and reduced opportunities for favorable tertiary interactions, Ca(2+)-free rat beta-parvalbumin is more stable than rat alpha-parvalbumin. Under conditions wherein alpha denatures at 45.8 degrees C, beta denatures at 53.6 degrees. The homologous chicken beta isoform known as CPV3 also exhibits heightened stability-prompting an inquiry into the stabilizing influence of Pro-21 and Pro-26. Individual P21A and P26A mutations lower the T(m) of rat beta by 3.2 degrees, decreasing conformational stability by 0.74 kcal/mol. Simultaneous replacement of Pro-21 and Pro-26 essentially abolishes the excess stability (DeltaT(m) = -7.6 degrees; DeltaDeltaG(conf) = -1.77 kcal/mol). Significantly, the P21A/P26A variant displays Ca(2+) affinity virtually indistinguishable from wild-type beta, implying that structural alterations in the AB domain do not necessarily influence the divalent ion affinity of the CD-EF domain. The consequences of introducing proline at positions 21 and 26 in rat alpha were also examined. Whereas the H26P mutation raises the T(m) by 5.6 degrees (DeltaDeltaG(conf) = 1.25 kcal/mol), A21P lowers the T(m) by 8.5 degrees (DeltaDeltaG(conf) = -1.9 kcal/mol). Replacement of Ala-21 by proline in an alpha AB/beta CD-EF chimera increases the T(m) by 5.8 degrees (DeltaDeltaG(conf) = 0.95 kcal/mol), implying that the destabilization of alpha by Pro-21 results from steric conflict with a residue in the CD-EF domain. Consistent with that hypothesis, the K80S mutation markedly stabilizes alpha A21P, yielding a protein with a T(m) 2.0 degrees higher than wild-type alpha. The observed differences in stability resulting from proline addition/removal are largely consistent with alterations in main-chain and side-chain conformational entropy.     

Transient high-affinity binding of agonists to alpha 1-adrenergic receptors of intact liver cells

At alpha 1-adrenergic receptors in isolated rat liver parenchymal cells, (-)-epinephrine is potent in eliciting a maximal increase in glycogenolysis (Kact = 24 nM). This contrasts with a 100-fold lower affinity for the agonist at alpha 1-adrenergic receptors of intact hepatocytes determined from equilibrium competition assays with the alpha 1-adrenergic antagonist [3H]prazosin. We demonstrate here that agonists bind to alpha 1-adrenergic receptors of intact liver cells initially with a markedly higher affinity than under equilibrium conditions. When incubations are performed for 15 s at 37 degrees C, the affinity is more than 100-fold higher than that obtained in equilibrium (45 min) assays (IC50 = 28 +/- 3 vs 5300 +/- 400 nM for (-)-epinephrine and 32 +/- 3 vs 6100 +/- 500 nM for (-)-norepinephrine). When incubations are performed at 4 degrees C (150 min), high-affinity binding similar to that obtained in short-term incubations can also be demonstrated. In contrast, antagonist compete with similar affinities in 15 s and 45 min assays, and their dissociation constants are not affected by changes in the incubation temperature. These results indicate that agonists bind to native alpha 1-adrenergic receptors transiently with high affinity. The conversion of receptors to a state of predominantly low affinity for agonists, which occurs rapidly and irreversibly with increasing incubation at 37 degrees C, is inhibited at low incubation temperatures. It is suggested that the high-affinity configuration of the alpha 1-adrenergic receptor for agonists observed in nonequilibrium experiments or at reduced incubation temperatures represents the physiologically relevant state of the alpha 1-adrenergic receptor.     

Alpha-amidated peptides derived from pro-opiomelanocortin in normal human pituitary.

Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well as their glycine-extended precursors, were characterized by sequence-specific radioimmunoassays, gel-chromatography, h.p.l.c. and amino acid sequencing. alpha MSH and gamma 1MSH constituted 0.27-1.32% and 0.10-5.10%, respectively, of the POMC-derived products [calculated as the sum of adrenocorticotropic hormone (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders of magnitude greater than alpha MSH and gamma 1MSH. Most (99%) of the HP-N was of low molecular mass and consisted mainly of HP-N-30. The remaining part was high-molecular-mass HP-N, probably HP-N-108, although the presence of HP-N-44 could not be completely excluded. These results show that all the possible amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests that the generation of amidated peptides is mainly regulated at the endopeptidase level.     

Characterization, size estimation and solubilization of alpha-macroglobulin complex receptors in liver membranes

Receptors for alpha 2-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of 125I-labelled alpha 2-macroglobulin.trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8-9.0. The half-time for association was about 5 min at 37 degrees C in contrast to about 5 h at 4 degrees C. The half-saturation constant was about 100 pM at 4 degrees C and 1 nM at 37 degrees C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 +/- 71 kDa (S.D., n = 7) for alpha 2-macroglobulin.trypsin binding activity. Affinity cross-linking to rat and human membranes of 125I-labelled rat alpha 1-inhibitor-3.chymotrypsin, a 210 kDa analogue which binds to the alpha 2-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55-60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked alpha 2-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound 125I-alpha 1-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400-500 kDa alpha 2-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos

Although fracture damage to the zonae pellucidae and blastomeres is frequently observed after the cryopreservation of mammalian embryos, little is known of the mechanism by which this occurs. The incidence of damage to zonae was measured when bovine ova with normal zonae were frozen in straws or glass test tubes by standard embryo cryopreservation procedures that yield high rates of survival. Ova were examined for zona damage after warming by procedures that ought to produce little or no thermal stress (slow warming in 20 degrees C air) or high levels of stress (rapid warming in liquid baths). Ova frozen in straws exhibited no zona damage after slow warming at 150 degrees C/min in air (n = 206). However, the incidence of zona damage increased when the straws were warmed rapidly in 20 degrees C (n = 157) or 36 degrees C (n = 159) water (17 and 24%, respectively). Ova in straws warmed rapidly in nonaqueous liquids (ethylene glycol, or silicone oil) exhibited lower rates of zona damage (2 to 5%). Ova frozen in glass tubes exhibited a much higher incidence of zona damage than those frozen in straws, regardless of the warming conditions. Thus, 30% of 114 ova exhibited damage when tubes were warmed slowly at 25 degrees C/min in air, while 54% of 98 ova showed zona damage when tubes were warmed rapidly at 500 degrees C/min in 36 degrees C water. These results are consistent with the view that zona damage is associated with thermally-induced fracturing of the suspension during rapid changes of temperature.     

Nitric oxide effects on force-velocity characteristics of the rat diaphragm

The present experiments tested nitric oxide (NO) effects on shortening velocity and power production in maximally activated rat diaphragm at 37 degrees C. Diaphragm fiber bundles (n = 10/group) were incubated at 37 degrees C in Krebs-Ringer solution containing no added drug (control), the NO synthase inhibitor Nomega-nitro-L-arginine (L-NNA; 10 mM), the NO donor sodium nitroprusside (SNP; 1 mM), or a combination (L-NNA + SNP). Loaded shortening velocity was measured via the load-clamp technique over a range of afterloads. Unloaded shortening velocity (Vo) was measured in control and L-NNA-treated bundles (n = 12/group) by using the slack test. Force-velocity data fitted to the Hill equation determined a Vmax of 13.7+/-0.4 lengths/s, contradicting the notion that rat diaphragm Vmax declines at temperatures > 35 degrees C. In contrast, L-NNA decreased Vmax (P < 0.05), loaded shortening velocity (P < 0.001), and power production (P < 0.001), but did not change Vo or maximal isometric force. All L-NNA effects were prevented by coincubating fiber bundles with L-NNA + SNP. SNP alone had no effect on any variable. These data indicate that endogenous NO is essential for optimal myofilament function during active shortening.     

Anomeric specificity of mannose phosphorylation by hexokinase

In rat parotid or pancreatic islet homogenates incubated at 7 degrees C, hexokinase displayed a greater affinity for but a lower maximal velocity with the alpha-anomer, as distinct from beta-anomer, of D-mannose. The anomeric specificity of mammalian hexokinase was similar in the case of D-mannose and D-glucose, but represented a mirror image of that of yeast hexokinase.     

Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B. subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores). Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C. After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations. However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb. These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.     

The effect of thawing velocity on survival and acrosomal integrity of ram spermatozoa frozen at optimal and suboptimal rates in straws

The effect of various thawing velocities on the motility and acrosomal maintenance of ram spermatozoa frozen at 20 degrees C/min (optimal) or 2 degrees C/min (suboptimal) was studied. The freeze-thaw motility and the percentage of intact acrosomes of spermatozoa frozen at 20 degrees C/min increased progressively with the thawing velocity. In semen frozen at 2 degrees C/min, motility of spermatozoa and the percentage of intact acrosomes declined drastically when the thawing velocity obtained in air at 20 degrees C was increased by thawing in water at 20 degrees C. Thawing at higher temperatures markedly increased both motility and acrosomal preservation, but the best results with semen frozen at 2 degrees C/min were lower than those obtained with semen frozen at 20 degrees C/min. The optimal freeze-thaw conditions for semen protected by 4% glycerol were freezing at 20 degrees C/min and thawing in water at 60 or 80 degrees C for 8 or 5 sec, respectively. Semen collected from rams exposed to a decreasing photoperiod exhibited higher motility after freezing and thawing than those exposed to an increasing photoperiod. However, there was no effect on acrosomal preservation after freezing at 20 degrees C/min.     

Analysis of nuclear RNA processing and transport by temperature perturbation of a cell-free system from mammalian cells

The similarity of the Arrhenius plots relating temperature to messenger RNA (mRNA) transport from intact and membrane-denuded rat liver nuclei demonstrates that the ATP and cytosol-dependent transport is independent of the lipid phase of the nuclear membrane. This temperature dependence of RNA release was confirmed for alpha 2u-globulin mRNA by use of a recombinant DNA probe. Ribosomal RNA (rRNA) release showed a similar temperature dependence, suggesting that both mRNA and rRNA share a common temperature-sensitive step. The kinetics of RNA release at different temperatures suggest that RNA transport from mammalian cell nuclei is a rate-controlled rather than a graded unlocking phenomenon. The processing of mRNA precursors also exhibits a temperature dependence as shown by the linear increase in the ratio of total alpha 2u-globulin RNA to alpha 2u-globulin precursor as a function of time at 30 degrees C but not at 14 degrees C in spite of residual transport at the lower temperature. This temperature dependence of mRNA processing was confirmed by Northern blot analysis of the nuclear RNA following a 45 min incubation. Thus, both the processing and transport of RNA show temperature-sensitive steps when analyzed in cell-free systems derived from mammalian cells.     

Inhibition of cell surface receptor-bound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin.

The purpose of this investigation was to characterize the reaction of alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M) with human plasmin bound to rat C6 glioma cells and human umbilical vein endothelial cells (HUVECs). Binding of plasmin (0.1 microM) to C6 cells at 4 degrees C did not cause cell detachment, decrease viability or change cell morphology. The KD and Bmax for the binding of diisopropyl phosphoryl plasmin (DIP-plasmin) to C6 cells were 0.9 microM and 2.6 x 10(6) sites/cell. The dissociation rate constants (koff) for 125I-plasmin were 9.7 x 10(-4) and 4.0 x 10(-4) s-1 at 4 degrees C in the presence and absence of 0.3 microM DIP-plasmin, respectively. Similar constants were determined for 125I-plasminogen and 125I-DIP-plasmin. Neither alpha 2AP nor alpha 2M affected the dissociation of DIP-plasmin. C6 cell-associated 125I-plasmin reacted slowly with alpha 2AP; however, the inhibition rate constants exceeded the koff. alpha 2AP-plasmin complex formed after the plasmin dissociated into solution (reaction pathway 1) and by direct reaction of alpha 2AP with cell-associated enzyme (reaction pathway 2). High concentrations of alpha 2AP favored pathway 2. C6 cell-associated plasmin was also protected from inhibition by alpha 2M. While the same pathways were probably involved in this reaction, alpha 2M was less effective than alpha 2AP as an inhibitor of nondissociated plasmin (pathway 2). When C6 cell-bound plasmin reacted with alpha 2AP, alpha 2AP-plasmin complex was recovered primarily in the medium, suggesting dissociation of complexes formed on the cell surface. Plasmin-receptor dissociation and inhibition experiments were performed at 22 degrees and 37 degrees C, confirming the conclusions of the 4 degrees C studies. Comparable results were also obtained using HUVEC cultures. These studies demonstrate that cell-associated plasmin is protected from inhibition by alpha 2M as well as alpha 2AP. At least two reaction pathways may be demonstrated for the inhibition of plasmin that is initially receptor-bound; however, neither pathway is highly effective, accounting for the "plasmin-protective" activity of the cell surface.     

Purification of the human alpha2 Isoform of Na,K-ATPase expressed in Pichia pastoris. Stabilization by lipids and FXYD1

Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state. alpha2beta1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha1beta1, while values of K0.5Na and KmATP are similar. Ouabain inhibits both alpha1beta1 (K0.5 24.6 +/- 6 nM) and alpha2beta1 (K0.5 102 +/- 14 nM) with high affinity. A striking difference between the isoforms is that alpha2beta1 is unstable. Both alpha1beta1 and alpha2beta1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha2beta1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (SOPS) stabilizes strongly, maintaining alpha2beta1 active up to two weeks at 0 degrees C. By contrast, alpha1beta1 is stable at 0 degrees C without added cholesterol. Both alpha1beta1 and alpha2beta1 complexes are stabilized by cholesterol at 37 degrees C. Human FXYD1 spontaneously associates in vitro with either alpha1beta1 or alpha2beta1, to form alpha1beta1/FXYD1 and alpha2beta1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha1beta1 and alpha2beta1 very strongly against thermal inactivation. Instability of alpha2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alphabeta subunit interface, may play an important role in differential interactions of lipid with alpha1 and alpha2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed.     

Isolation and characterization of pepsin-treated type III collagen from calf skin.

Calf skin collagen was solubilized by incubating acid-extracted calf skin with pepsin at pH 2.0 and 25 degrees C, conditions that did not cause degradation of the triple helical region of collagen. Type III collagen was separated from type I collagen by differential salt precipitation at pH 7.5. The isolated type III collagen contained mainly gamma and higher molecular weight components cross-linked by reducible and/or non-reducible bonds. The isolated alpha1 (III) chains had an amino acid composition characteristic of type III collagen. Denatured but unreduced type III collagen, chromatographed on carboxymethyl-cellulose, eluted in the alpha 2 region, while after reduction and alkylation the alpha1 (III) chains eluted between the positions of alpha1 (I) and alpha2. The mid-point melting temperature temperature (tm) of type III collagen (35.1 degrees C) in a citrate buffer at pH 3.7 was somewhat lower than that of type I collagen (35.9 degrees C). Renaturation experiments at 25 degrees C showed that denatured type III collagen molecules with intact intramolecular disulfide bridges (gamma components) reform the triple helical structure of collagen much faster than reduced and carboxymethylated alpha1 (III) chains.